U.S. patent application number 13/201927 was filed with the patent office on 2012-02-16 for sensing device for detecting a target substance.
This patent application is currently assigned to KONINKLIJKE PHILIPS ELECTRONICS N.V.. Invention is credited to Dominique Maria Bruls, Toon Hendrik Evers, Johannes Joseph Hubertina Barbara Schleipen.
Application Number | 20120040475 13/201927 |
Document ID | / |
Family ID | 40848456 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040475 |
Kind Code |
A1 |
Bruls; Dominique Maria ; et
al. |
February 16, 2012 |
SENSING DEVICE FOR DETECTING A TARGET SUBSTANCE
Abstract
The present invention relates to a sensing device (100) for
detecting a target substance (2) in an investigation region (113).
The sensing device (100) comprises a sensing surface (112) with an
investigation region (113) and a reference region (120) thereon.
The sensing device (100) further comprises a reference element
(121) located at the reference region (120). The reference element
(121) is adapted to shield the reference region (120) from the
target substance (2) such that light reflected at the reference
region (120) under total internal reflection conditions remains
unaffected by the presence or absence of the target substance (2).
This allows measuring a property, typically the intensity, of light
reflected at the reference region (120) independent of the presence
or absence of the target substance (2). This measured property of
the reflected light can be used for performing an improved
correction of light reflected at the investigation region
(113).
Inventors: |
Bruls; Dominique Maria;
(Heeze, NL) ; Evers; Toon Hendrik; (Eindhoven,
NL) ; Schleipen; Johannes Joseph Hubertina Barbara;
(Eindhoven, NL) |
Assignee: |
KONINKLIJKE PHILIPS ELECTRONICS
N.V.
EINDHOVEN
NL
|
Family ID: |
40848456 |
Appl. No.: |
13/201927 |
Filed: |
February 8, 2010 |
PCT Filed: |
February 8, 2010 |
PCT NO: |
PCT/IB2010/050553 |
371 Date: |
October 27, 2011 |
Current U.S.
Class: |
436/501 ;
356/445; 422/69 |
Current CPC
Class: |
G01N 33/54373 20130101;
G01N 21/274 20130101; G01N 21/552 20130101 |
Class at
Publication: |
436/501 ;
356/445; 422/69 |
International
Class: |
G01N 21/55 20060101
G01N021/55 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 2009 |
EP |
09153105.3 |
Claims
1. A sensing device (100) for detecting a target substance (2) in
an investigation region (113), comprising a light source (11)
generating an incident light beam L1, a sensing surface (112) with
an investigation region (113) and a reference region (120), a
reference element (121) located at the reference region (120)
adapted to shield the reference region (120) from the target
substance (2) such that light reflected at the reference region
(120) under total internal reflection conditions remains unaffected
by the presence or absence of the target substance (2), a
calibrator (20) comparing the outgoing light L2 of both the
reference region (120) and the investigation region (113). wherein
the reference element (121) has such refractive index and such
dimensions that an evanescent field elicited at the reference
region (120) remains unaffected by the presence or absence of the
target substance (2).
2. The sensing device (100) according to claim 1, wherein the
investigation region (113) comprises a binder (114) for binding the
target substance (2).
3. The sensing device (100) according to claim 2, wherein the
sensing device (100) is adapted to analyse the presence of a
substance (2, 2') in a medium (4) at a concentration of less than
or equal to 1 nM.
4. The sensing device (100) according to claim 1, wherein the
sensing surface (112) at the reference region (120) is tilted
relatively to the sensing surface (112) of the investigation region
(113) to allow incidence of an incident light beam (L1) at the
reference region (120) at an angle shallower than that of a
parallel incident light beam at the investigation region (113).
5. The sensing device (100) according to claim 1, wherein the
reference region (120) is adjacent to the investigation region
(113).
6. The sensing device (100) according to claim 1, wherein the
reference region (120) comprises a mirror (121) to reflect incident
light.
7. The sensing device (100) according to claim 1, wherein the
sensing device (100) is a cartridge (100) having a carrier (110)
comprising the sensing surface (112) thereon.
8. The sensing device (100) according to claim 1, further
comprising a light source (11) to direct incident light (L1) to the
investigation region (113) and the reference region (120) of the
sensing surface (112) such that the incident light (L1) is
reflected under total internal reflection conditions at the
investigation region (113) and the reference region (120) thereby
generating reflected light (L2), a detector (18) for detecting the
reflected light (L2) to yield a first characteristic signal (213)
depending on the reflection at the investigation region (113) and a
second characteristic signal (220) depending on the reflection at
the reference region (120), and a calibrator (20) for calibrating
the first characteristic signal (213) in view of the second
characteristic signal (220).
9. The sensing device (100) according to claim 8, wherein the
calibrator (20) is adapted to correct or ameliorate a drift in the
first characteristic signal (213) in view of the second
characteristic signal (220).
10. The sensing device (100) according to claim 1, adapted to
cooperate with an analyzing device for detecting the target
substance (2), wherein the analyzing device comprises: an analysis
region for accommodating the sensing device (100), a light source
(11) to direct incident light (L1) to the analysis region such that
the incident light (L1) is directed to the investigation region
(113) and the reference region (120) of the sensing surface (112)
and such that the incident light (L1) is reflected under total
internal reflection conditions at the investigation region (113)
and the reference region (120), thereby generating reflected light
(L2), when the sensing device (100) is accommodated in the analysis
region, a detector (18) for detecting the reflected light (L2) to
yield a first characteristic signal (213) depending on the
reflection at the investigation region (113) and a second
characteristic signal (220) depending on the reflection at the
reference region (120), a calibrator (20) for calibrating the first
characteristic signal (213) in view of the second characteristic
signal (220).
11. An analyzer device (10) for detecting a target substance (2),
adapted to cooperate with a sensing device (100) for detecting the
target substance (2), wherein the sensing device (100) comprises: a
sensing surface (112) with an investigation region (113) and a
reference region (120) thereon, a reference element (121) located
at the reference region (120) adapted to shield the reference
region (120) from the target substance (2) such that light
reflected at the reference region (120) under total internal
reflection conditions remains unaffected by the presence or absence
of the target substance (2), wherein the analyzer device (10)
comprises: an analysis region for accommodating the sensing device
(100), a light source (11) to direct incident light (L1) to the
analysis region such that the incident light (L1) is directed to
the investigation region (113) and the reference region (120) of
the sensing surface (112) and such that the incident light (L1) is
reflected under total internal reflection conditions at the
investigation region (113) and the reference region (120), thereby
generating reflected light (L2), when the sensing device (100) is
accommodated in the analysis region, a detector (18) for detecting
the reflected light (L2) to yield a first characteristic signal
(213) depending on the reflection at the investigation region (113)
and a second characteristic signal (220) depending on the
reflection at the reference region (120), a calibrator (20) for
calibrating the first characteristic signal (213) in view of the
second characteristic signal (220).
12. The analyzer device (10) according to claim 11, wherein the
calibrator (20) is adapted to correct or ameliorate a drift in the
first characteristic signal (213) in view of the second
characteristic signal (220).
13. A sensing method for detecting a target substance (2) in an
investigation region (113), comprising the steps of providing a
sensing surface (112) with an investigation region (113) and a
reference region (120) thereon, providing a reference element (121)
located at the reference region (120) adapted to shield the
reference region (120) from the target substance (2) such that
light reflected at the reference region (120) under total internal
reflection conditions remains unaffected by the presence or absence
of the target substance (2), illuminating the investigation region
(113) and the reference region (120) under total internal
reflection conditions.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a sensing device and an
analyzing device for sensing a target substance in an investigation
region. The invention further relates to a corresponding sensing
method for sensing a target substance in an investigation
region.
BACKGROUND OF THE INVENTION
[0002] U.S. Pat. No. 7,317,534 B2 provides a measuring method
comprising a measuring unit with a film layer having a detecting
area where a target molecule is fixed to the surface thereof and a
reference area where no ligand is fixed to the surface thereof. A
photo detector detects the intensities of light beams reflected in
total internal reflection at the detecting area and reference area,
respectively. Further, the result of measurement in the detecting
area is calibrated on the bases of the result of measurement in the
reference area.
[0003] US2005/0052655 A1 describes an interferometer comprising an
optical body adapted in operation to mount a measurement area
comprising a film which is capable of acting as a two dimensional
environment for surface plasmons and an adjacent reference area, an
optical beam generation means for irradiating the reference and
measurement areas with radiation capable of generating surface
plasmon resonance, optical means for combining radiation reflected
from the reference and measurement areas, and pixelated detection
means for generating data representing two dimensional images of
the combined radiation beams.
SUMMARY OF THE INVENTION
[0004] However, it is frequently difficult to guarantee that a
reference area remains free of any target molecule or otherwise
unaffected by the presence of the target molecule in the medium.
Particularly when employing bead-based target molecule detection
systems, it is difficult to ascertain that no such beads
unspecifically come into contact with the reference area and
influence the signal detected from the reference area. Thus,
measures must be provided for ensuring that the reference area
remains free of any target molecule during calibration, which
renders the calibration procedure quite complicate.
[0005] It is an object of the invention to provide a sensing device
and an analyzing device for detecting a target substance in an
investigation region that allow for easier calibration. It is a
further object of the present invention to provide a corresponding
sensing method.
[0006] In an aspect of the present invention a sensing device for
detecting a target substance in an investigation region is
provided, comprising [0007] a sensing surface with an investigation
region and a reference region thereon, [0008] a reference element
located at the reference region adapted to shield the reference
region from the substance such that light reflected at the
reference region under total internal reflection conditions remains
unaffected by the presence or absence of the substance.
[0009] The invention is based on the idea that a signal
corresponding to the presence of a substance at the investigation
region obtained by detecting light reflected from the investigation
region under total internal reflection conditions can be calibrated
by a signal corresponding to a standard reflecting light under
total internal reflection conditions. Thus, a reference element
effectively shielding the reference region from the influence of
substances potentially present at the investigation region allows
to measure a property, typically the intensity, of light reflected
at the reference region in a standardized form, i.e. not dependent
on the presence or absence of the substance to be analysed. Thus,
the light reflected at the reference region is substantially only a
function of factors other than the presence or absence of the
substance and thus reflects errors due to measurement errors, e.g.
increases in temperature or fluctuations in the light output
reaching the reference region.
[0010] Within the present invention, light reflected at the
reference region under total internal reflection conditions is
considered unaffected by the presence or absence of the substance
when a selected property of the light, typically its intensity, is
not altered by the presence or absence of the substance by more
than 10%, preferably not more than 1%, and most preferably not more
than 0.1%. Particularly preferred is a range of 0.05 to 0.3%.
[0011] Further preferably, the reference element shields the
reference region not only from the substance to be analysed, but
also against other substances potentially influencing an evanescent
field present at the reference region. This advantageously allows
to analyse the presence of a substance in a coloured medium, as the
coloration of the medium would not interfere with the light
reflected at the reference region under total internal reflection
conditions. Thus, even for coloured media the reference region can
serve as a reliable standard. The method of the present invention
and the sensing devices of the present invention can also
advantageously be used to complement analytical methods involving
measurement of transmitted light, for example measurement of light
absorption and optical density. It is thus advisable for the
reference element to be preferably solid, gel-like or otherwise
resistant against being washed away by the medium.
[0012] It should be noted that the term "total internal reflection"
shall include the case frequently termed "frustrated total internal
reflection", where some of the incident light is lost during the
reflection process. The reflected light beam originating at the
investigation region will typically consist of or comprise light of
the incident light beam that was totally internally reflected at
the investigation region of the sensing surface, which might be a
binding surface for binding the target substance. It may, however,
also comprise light from other sources like a fluorescence
stimulated in the investigation region.
[0013] The refractive index and dimensions of the reference element
are preferably chosen such that the dimensions exceed the
exponential decay length of an evanescent field elicited therein,
i.e. such that the evanescent field is substantially not located
outside of the reference element and of a carrier on which the
reference element is located. The thickness and lateral dimensions
of the reference element are then chosen in such a way that the
evanescent field generated under total internal reflection
conditions inside the reference element is substantially damped out
for a chosen wavelength and angle of incidence of light. The
exponential decay length .zeta. scales as
.zeta. = .lamda. n 1 2 .pi. sin 2 ( .theta. ) - sin 2 ( .theta.
crit ) with ( 1 ) .theta. crit = sin - 1 ( n 2 n 1 ) , ( 2 )
##EQU00001##
[0014] wherein .lamda., and .theta. are the wavelength of the used
light and the angle of incidence, respectively, and n.sub.1 and
n.sub.2 are the refractive indices of the material the light
travels in and of the reference element, respectively. The angle
.theta..sub.crit defines the critical angle. Thus, the skilled
person can select suitable materials for manufacturing of the
reference element for a preselected wavelength, angle of incidence
and material for the light ray to travel in.
[0015] The lateral shape of the reference element can be chosen
arbitrarily, thus allowing the present device to be adapted to a
plethora of ancillary conditions. Preferably, the reference element
is a film or layer having a thickness of at least 200 nm, more
preferably 500 nm-1 mm and most preferably 500 nm-100 .mu.m. Such
elements advantageously can be included in microsensors allowing to
analyse minute quantities of a medium.
[0016] In a further preferred sensing device, the investigation
region comprises a binder for binding a target. The target can be a
target substance which can influence an evanescent field on its
own. However, in case the substance whose concentration is to be
determined requires a label to influence an evanescent field for
its detection--hereinafter termed "analyte"--, the target would be
such label. Typically, the target will then be a substance,
preferably a magnetic bead, comprising a coupling section to attach
to the analyte, preferably by an attachment specific for the
analyte in view of other substances that are expected to be further
comprised in the medium. The coupling section may be linked
covalently or non-covalently to the analyte and the rest of the
target substance, respectively. Preferably, the target comprises
one, two, three or more antibodies or Fab fragments thereof,
including F(ab').sub.2 fragments, which can bind to the analyte at
the same or at different sections of the analyte.
[0017] A typical example of a binder of the investigation region is
an antibody or Fab fragment thereof, including F(ab').sub.2
fragments. Such binders can be produced for a huge variety of
target molecules and antigens thereof and allow for the specific
binding of targets and/or analytes, where applicable, at the
investigation region of the sensing surface. It is thus possible to
selectively enrich these targets/analytes at the investigation
region. Moreover, undesired targets can be removed from the
investigation region by suitable repelling forces (e.g. magnetic or
hydrodynamic forces) that should not substantially break the
binding between desired target molecules and binders. Binding of
the target may influence the evanescent field elicited at the
investigation region and thus influence the intensity of light
reflected therefrom under total internal reflection conditions.
Also further substances may be added to augment the influence
exerted by a bound target at the investigation region on the
evanescent field elicited thereon. Further, the investigation
region may comprise one, two or more types of binders. The types of
binders can be specific for different target molecules or for
different sections and antigens of one or more target molecules.
The sensing surface of the present invention is thus suitable for
different kinds of bio sensors and measuring methods.
[0018] Another type of assay for determining the concentration of
an analyte in a medium can be a competitive binding assay. In such
assay, quantitation of an analyte concentration can be achieved by
analysing the presence or absence of a target at a respective
investigation region as a result of a competition between the
analyte and an analyte-like substance for binding by the binder
and/or the target or its respective coupling section.
[0019] The sensing device of the present invention is preferably
adapted to analyse the presence of a substance (i.e. target or, if
applicable, analyte) in a medium at a concentration of less than or
equal to 1 nM, even more preferably from 1 to 1000 pM and most
preferably from 10 to 1000 fM. Such low concentrations typically
require long measuring times and the obtained signals from the
medium are small as well. The signals generated by typical optical
sensor devices can drift over time with no changes in assay
composition. For example, the light output of the light source may
vary, e.g. due to temperature changes of the environment or within
the biosensor device. The drift can lead to significant deviations
of the obtained signal compared to the real amount of target
molecules bound at the investigation region. Thus, a calibration of
the signal obtained by the detector from the investigation region
is necessary to obtain significant results. It is a particularly
valuable advantage of the present invention to allow such
calibration based on the reference region and light reflected
thereat, such allowing to reliably analyse the presence or absence
of a substance at the low concentrations mentioned before. This is
particularly advantageous when measuring the presence of substances
like e.g. cardiac Troponin-I, parathyroid hormone (PTH) and BNP
(brain natriuretic peptide), in physiological samples, e.g. blood,
which require detection limits of less than 1 pM.
[0020] In a preferred sensing device the sensing surface at the
reference region is tilted relatively to the sensing surface of an
investigation region to allow incidence of an incident light beam
at the reference region at an angle shallower than that of a
parallel incident light beam at the investigation region. To
achieve total internal reflection conditions at the sensing
surface-reference element interface, the refractive index of the
reference element material must be chosen low enough for a given
refractive index of the material the reflected light travels in.
For some materials it is thus difficult to find a suitable
reference element material. By slightly tilting the surface at the
reference region, it is possible achieve an angle of incidence
shallower at the reference element than at an investigation area,
thus allowing to use reference element materials with comparatively
higher refractive index.
[0021] In a further preferred sensing device, the reference region
is preferably adjacent to the investigation region. This way,
errors in intensity measurements of light reflected under total
internal reflection conditions e.g. due to slight variations in the
carrier material compositions can be minimized. Within the present
invention, a reference region is considered adjacent when it is
separated from an investigation region, preferably an investigation
region having attached a binder thereto as described above, by at
most 5 mm, more preferably 0.5 to 1 mm, and most preferably 0.1 to
0.5 mm.
[0022] The material of the reference element can be chosen
arbitrarily, as long as it allows total internal reflection at the
reference region for a preselected wavelength and angle of
incidence of light and sufficiently shields the reference region as
indicated above. Preferably, the reference element is a solid, even
more preferably the reference element is a film. Within the present
invention, glasses are considered solids. Further preferred
materials of the reference element are chosen from polymers,
biomolecules and particularly proteins, nucleic acids and
polysaccharides, gels, sol-gels and other plastics.
[0023] It is preferred to choose a material that can be deposited
in a simple way, e.g. ink-jet printing. Many polymers are available
that can be UV-cured or be polymerized by other suitable means for
fast, reliable and automated polymerization.
[0024] It is further preferred that the reference region comprises
a mirror to reflect incident light. Such mirror, frequently termed
true mirror, can be applied in the form of a dielectric multilayer
or metallic coating and is particularly suitable as a reference
element in such conditions where finding a reference element
material with sufficiently low refractive index is difficult.
[0025] In many practically relevant embodiments of the sensing
device, the sensing surface will comprise two or more investigation
regions at which different incident light beams can be totally
internally reflected. One device then allows the processing of
several investigation regions and thus for example the search for
different target substances, the observation of the same target
substances under different conditions and/or the sampling of
several measurements for statistical purposes. The "different
incident light beams" may optionally be components of one broad
light beam that is homogeneously generated by one light source,
they may be individual separate light beams addressing the
investigation regions and/or reference regions simultaneously
(optionally through the same or through different optical windows),
and/or they may be temporally different (i.e. be generated by one
generic light beam scanning the investigation regions). Preferably,
the "different incident light beams" are part of one broad light
beam simultaneously illuminating the one, two or more reference
region(s) and the one, two or more investigation region(s). A
detector may then measure the light reflected from the respective
regions separately, als will be described in greater detail
below.
[0026] The sensing device preferably is a cartridge having a
carrier comprising the sensing surface thereon. Such cartridges can
be advantageously used with analyzing devices, to adapt the
analyzing devices to specific measurement tasks.
[0027] While it is in principle possible that the carrier has some
dedicated structure with multiple components of different
materials, it is preferred that the carrier is homogenously
fabricated from a transparent material, for example from glass or a
transparent plastic. The carrier can thus readily be produced for
example by injection moulding.
[0028] The cartridge may be used in combination with many different
devices including biosensor devices and methods. For a practically
important application in an investigation procedure, the cartridge
preferably comprises a first and a second optical window, such that
an incident light beam can enter the carrier through the first
optical window such that it is totally internally reflected at the
investigation region and/or the reference region at the sensing
surface, and wherein a reflected light beam originating at the
investigation region and/or the reference region can exit the
carrier through the second optical window.
[0029] The sensing device of the present invention preferably
further comprises [0030] a light source to direct incident light to
the investigation region and the reference region of the sensing
surface such that the incident light is reflected under total
internal reflection conditions at the investigation region and the
reference region thereby generating reflected light, [0031] a
detector for detecting the reflected light to yield a first
characteristic signal depending on the reflection at the
investigation region and a second characteristic signal depending
on the reflection at the reference region, and [0032] a calibrator
for calibrating the first characteristic signal in view of the
second characteristic signal.
[0033] Such device makes use of the advantage of the present
invention, i.e. the sensing device allows to calibrate the first
characteristic signal in view of the second characteristic signal.
The sensing device thus particularly facilitates reliable,
significant measurements, limits measurement errors and allows
detecting a target substance at the investigation region(s) at low
concentrations by enabling long(er) measurement times.
[0034] In practical embodiments of such device, the calibrator is
preferably adapted to correct or ameliorate a drift in the first
characteristic signal in view of the second characteristic signal.
It is an advantage of such device that particularly long
measurement times without significant influence of drift can be
achieved, thus facilitating or even enabling to detect target
substances at low concentrations at an investigation region or
against a noisy medium background.
[0035] In a preferred embodiment, the sensing device is adapted to
cooperate with an analyzing device for detecting the target
substance, wherein the analyzing device comprises: [0036] an
analysis region for accommodating the sensing device, [0037] a
light source to direct incident light to the analysis region such
that the incident light is directed to the investigation region and
the reference region of the sensing surface and such that the
incident light is reflected under total internal reflection
conditions at the investigation region and the reference region,
thereby generating reflected light, when the sensing device is
accommodated in the analysis region, [0038] a detector for
detecting the reflected light to yield a first characteristic
signal depending on the reflection at the investigation region and
a second characteristic signal depending on the reflection at the
reference region, [0039] a calibrator for calibrating the first
characteristic signal in view of the second characteristic
signal.
[0040] In a further aspect of the present invention there is
provided an analyzer device for detecting a target substance,
adapted to cooperate with a sensing device for detecting the target
substance, wherein the sensing device comprises: [0041] a sensing
surface with an investigation region and a reference region
thereon, [0042] a reference element located at the reference region
adapted to shield the reference region from the target substance
such that light reflected at the reference region under total
internal reflection conditions remains unaffected by the presence
or absence of the target substance,
[0043] wherein the analyzing device comprises: [0044] an analysis
region for accommodating the sensing device, [0045] a light source
to direct incident light to the analysis region such that the
incident light is directed to the investigation region and the
reference region of the sensing surface and such that the incident
light is reflected under total internal reflection conditions at
the investigation region and the reference region, thereby
generating reflected light, when the sensing device is accommodated
in the analysis region, [0046] a detector for detecting the
reflected light to yield a first characteristic signal depending on
the reflection at the investigation region and a second
characteristic signal depending on the reflection at the reference
region, [0047] a calibrator for calibrating the first
characteristic signal in view of the second characteristic
signal.
[0048] Such an analyzer device is advantageously adapted to a
sensing device, particularly a cartridge, of the present invention
and makes use of the reference region for calibrating a
characteristic signal obtained from the investigation region. The
characteristic signal preferably is a light intensity signal. The
sensing device preferably is a biosensor.
[0049] Again, the calibrator is preferably adapted to correct or
ameliorate a drift in the first characteristic signal of light
emanating from an investigation region of the sensing device,
particularly a cartridge, in view of the second characteristic
signal of light emanating from a reference region of the sensing
device.
[0050] Thus, the analyzer device allows to reduce the measurement
error for light intensity measurements at the investigation region.
It is thus possible to analyze light reflected from the
investigation region for a long period of time without
significantly tainting the first characteristic signal obtained
from said investigation region by erroneous light intensity drifts.
Such analyzing device thus facilitates detection of substances at
an investigation region at the very low concentrations which
typically require a long irradiation time of the investigation
region.
[0051] According to a further aspect of the invention there is
provided a sensing method for detecting a target substance in an
investigation region, comprising the steps of [0052] providing a
sensing surface with an investigation region and a reference region
thereon, [0053] providing a reference element located at the
reference region adapted to shield the reference region from the
target substance such that light reflected at the reference region
under total internal reflection conditions remains unaffected by
the presence or absence of the target substance, [0054]
illuminating the investigation region and the reference region
under total internal reflection conditions.
[0055] The sensing method can further comprise the steps of:
[0056] a) irradiating an investigation region of a sensing device
of the invention with light and obtaining a first characteristic
signal of totally internally reflected light therefrom,
[0057] b) before, during or after step a) irradiating a reference
region of the sensing device of the present invention with light
and obtaining a second characteristic signal of totally internally
reflected light therefrom,
[0058] c) calibrating the first characteristic signal obtained in
step a) by the second characteristic signal obtained in step
b).
[0059] The sensing method allows to detect the presence of a target
substance at an investigation region, and also allows to determine
another property of the investigation region, e.g. to determine a
temperature by analysing the influence of a thermochromic substance
on the intensity of light reflected under total internal reflection
conditions at the investigation region.
[0060] Preferably, steps a) and c), steps b) and c) or steps a), b)
and c) are repeated. Such repetition can be performed for one
investigation region to allow a time-resolved analysis of target
substance binding at the investigation region. The steps can also
be repeated by applying them sequentially to different
investigation regions, allowing to determine the amount of target
substance(s) bound at different investigation regions.
[0061] It is particularly preferred to at least perform steps a)
and b) simultaneously for at least one reference region and one
investigation region. This way, the calibration can be performed
with very low measurement errors. Further preferred is a sensing
method, comprising
[0062] i) providing an analyzing device of the present
invention,
[0063] ii) providing a sensing device, preferably a cartridge, of
the present invention at an analysis region of said analyzing
device,
[0064] iii) binding a target substance at an investigation region
of said sensing device,
[0065] iv) performing the measuring method steps a), b) and c) as
described above.
[0066] Such method exploits the advantages of the present invention
and particularly allows analysis of a target substance at a very
low concentration at an investigation region as described
above.
[0067] It shall be understood that the sensing device of claim 1,
the analyzer device of claim 12 and the sensing method of claim 14
have similar and/or identical preferred embodiments as defined in
the dependent claims. It shall further be understood that a
preferred embodiment of the invention can also be any combination
of the dependent claims with the respective independent claim.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1 shows schematically and exemplarily a side view of an
embodiment of a sensing device,
[0069] FIG. 2 shows schematically and exemplarily a graph of a
signal drift of an uncorrected detection signal,
[0070] FIG. 3 shows schematically and exemplarily a side view of
light reflected under total internal reflection conditions,
[0071] FIG. 4 shows schematically and exemplarily a top view of an
embodiment of a sensing device,
[0072] FIG. 5 shows schematically and exemplarily a side view of
the embodiment of a sensing device of FIG. 4,
[0073] FIG. 6 shows schematically and exemplarily a top view of an
embodiment of a sensing device,
[0074] FIG. 7 shows schematically and exemplarily a side view of
the embodiment of a sensing device of FIG. 6,
[0075] FIG. 8 shows schematically and exemplarily a graph of a
detection signal and a reference signal,
[0076] FIG. 9 shows schematically and exemplarily a side view of an
embodiment of a sensing device,
[0077] FIG. 10 shows schematically and exemplarily a top view of
the embodiment of a sensing device of FIG. 9,
[0078] FIG. 11 shows schematically and exemplarily a side view of
an embodiment of a sensing device,
[0079] FIG. 12 shows schematically and exemplarily a side view of
an embodiment of an analyzing device,
[0080] FIG. 13 shows schematically and exemplarily magnetic
particles bound to an investigation region of a sensing device,
and
[0081] FIG. 14 shows schematically and exemplarily a method of the
present invention.
DETAILED DESCRIPTION OF EMBODIMENTS
[0082] FIG. 1 shows schematically and exemplarily a side view of an
embodiment of a sensing device 100 of the present invention. The
device 100 comprises a carrier 110 of a material transparent for an
incident light beam L1. The carrier 110 has a sensing surface 112.
On the sensing surface 112, a fluid delimiter 101 is positioned
such as to allow a medium 4 to be added to the sensing surface 112.
An area of the sensing surface 112 is covered by a reference
element 121 to shield a reference region 120 of the sensing surface
112 from any target substance (2) in the medium 4. In the vicinity
of the reference region 120 is an investigation region 113 of the
sensing surface 112. The investigation region 113 can comprise a
binder 114 for direct or indirect binding of the target substance
2.
[0083] Both the reference region 120 and the investigation region
113 can be illuminated by incident light (shown as incident light
beam L1). The incident light L1 is reflected under total internal
reflection conditions at the reference region 120 to produce
outgoing light L2. The reference element 121 has such refractive
index and such dimensions that an evanescent field elicited by
total internal reflection at the reference region 120 remains
unaffected by the presence or absence of the target substance 2.
Thus, the intensity of the outgoing light beam L2 of the reference
region 120 does not depend on the presence or absence of the target
substance 2 in the medium 4, and is preferably also independent of
the presence or absence of other substances of the medium 4. The
outgoing light beam L2 of the reference region 120 can then serve
as a standard light beam, and its intensity can be used as a
(second) characteristic signal 220 at a detector 18.
[0084] The incident light L1 is also reflected under total internal
reflection conditions at the investigation region 113 to produce
further outgoing light L2. However, an evanescent field elicited at
the investigation region 113 is subject to influences of the target
substance 2 of the medium 4, and most preferably only or mainly of
the target substance 2. The intensity of such further outgoing
light then correlates with the presence or absence of the target
substance 2 at the investigation region 113 and can serve as a
(first) characteristic signal 213 at the detector 18.
[0085] A calibrator 20 compares the outgoing light L2 of both the
reference region 120 and the investigation region 113. By such
comparison the influence of the target substance 2 on the intensity
of light reflected at the investigation region 113 under total
internal reflection conditions can be determined. This mode of
comparison reduces or eliminates any further sources of measurement
error, as the comparison can be made largely independent of the
variation in incident light L1 intensity by simultaneous or
sequentially swift measurement of the intensity of outgoing light
L2 of the reference region 120 and the investigation region 113.
Also, both first and second characteristic signal 213 and 220,
respectively, can be determined at a single detector 18, further
reducing measurement errors. The calibrator 20 thus effectively
corrects or ameliorates a drift in the first characteristic signal
213 in view of the second characteristic signal 220.
[0086] To produce a reference element 121, a small drop of
UV-curable, low refractive index acrylate
(2,2,3,3,4,4,5,5-Octafluoro-hexanedio1-1,6-dimethacrylate) can be
applied on a sensing surface 112 of a polystyrene cartridge. After
curing under nitrogen conditions, a reference element 121 with
n.sub.2<1.42 at a reference region 120 can thus be obtained. For
such reference element 121 it was found that when an adjacent
investigation region 113 of the sensing surface 112 was blackened
with a black marker pen, the intensity of light reflected (L2)
under total internal reflection conditions (angle of incidence
.theta..sub.i: 70.degree.) decreased markedly at the investigation
region 113. However, even though the reference element 121 was also
covered by ink of the black marker pen, the intensity of light
reflected under total internal reflection conditions (angle of
incidence .theta..sub.i: 70.degree.) did not decrease markedly at
the reference region 120, as shown in FIG. 8.
[0087] The refractive index n.sub.2 of the cured acrylate is still
rather high. Under the aforementioned special experimental
conditions, the incoming beam was not perfectly parallel, such that
part of the incident light had a smaller angle than 70.degree..
This means that a small portion of the incoming light beam
penetrated the reference element 121 and was thus subjected to the
influence of the black marker ink, reducing the intensity of
reflected light.
[0088] This can be solved by further reducing the refractive index
on the reference element 121 and/or by increasing the angle of
incidence and/or by increasing the refractive index of the
cartridge material and/or by improving the collimation of the
incoming illumination beam. Instead of a polymer, other low
refractive index materials may be suitable like e.g. biomolecules
(particularly proteins, nucleic acids, polysaccharides), gels,
sol-gels or other plastics. The way these different reference
elements 121 are applied to a sensing surface 112 is dependent on
the nature of the reference element 121, the sensing surface 112
and the cartridge material. For example, in case of biomolecules it
might be necessary to covalently attach them to the cartridge
sensing surface 112.
[0089] FIG. 2 shows schematically and exemplarily a graph of a
signal drift of an uncorrected detection signal. The x-axis refers
to measurement time t given in minutes. The y-axis refers to the
variation (given as a percentage) of a first characteristic signal
213 of light of an investigation region 113 of a sensing device 100
of the type of FIG. 1. No substance was added to the investigation
region 113 during the measurement of FIG. 2. The graph shows that
after 10 minutes of measuring time, the first characteristic signal
213 had drifted by 0.1%. Since this drift was not caused by the
addition of a substance to the investigation region 113, the drift
must be considered a measurement error. Without being bound to any
particular theory, such drift is considered to result from
arbitrary variations in the intensity of a light source 11, e.g. a
LED, and the sensitivity of a sensor, e.g. a CMOS sensor,
particularly due to temperature changes in the device. Such
measurement errors cannot be tolerated in analytical tasks
requiring high detection sensitivity and precision, e.g. the
determination of Troponin-I concentration in a medium 4 like blood.
Such analytical tasks can require a detection limit of less than 1
pM which, in turn, require long measuring times with very low
signal drift. The device of FIG. 1 now allows to reliably
accomplish these analytical tasks.
[0090] FIG. 3 shows schematically and exemplarily a side view of
light reflected under total internal reflection conditions and
illustrates the principle of total internal reflection. An incident
light beam L1 travels through a medium 4 with a first refractive
index n.sub.1. The light beam L1 reaches a surface to another
medium 4 with a second refractive index n.sub.2. The light beam L1
is then reflected under total internal reflection conditions to
form a light beam L2 if the angle of incidence .theta..sub.i is
larger than a critical angle .theta..sub.crit, where according to
Snell's law .theta..sub.crit=sin.sup.-1 (n.sub.2/n.sub.1).
[0091] For example, a material useful for manufacture of biosensor
cartridges is polystyrene. For a polystyrene material, n.sub.1 is
1.55. Further, when choosing an angle of incidence .theta..sub.i of
70.degree. as would be useful for a biosensor device having a
polystyrene/water-like sensor interface, the second medium 4 should
be chosen such that n.sub.2<1.45. .theta..sub.crit would then be
69.1.degree., such that total internal reflection would occur at
the surface between both media.
[0092] In preferred sensing devices, some margin is included for
n.sub.2, as there is frequently some angular distribution in the
angle of incidence, e.g. as a LED might not produce perfectly
parallel beams. To have an angular margin of 2.degree., n.sub.2
should be chosen to be less than 1.419.
[0093] FIGS. 4 and 5 schematically and exemplarily show a top and
side view of a sensing device 100, respectively. The sensing device
100 has a cartridge 110 of a first refractive index n.sub.1. The
cartridge 110 has a sensing surface 112. On the sensing surface
112, a reference element 121 is positioned to shield a reference
area 120 of the sensing surface 112. Adjacent to the reference area
120 is an investigation region 113 of the sensing surface 112. The
investigation region 113 does not comprise a reference element 121.
In FIG. 5, the investigation region 113 is obscured by the
reference element 121.
[0094] Both the investigation region 113 and the reference region
120 can be illuminated by a beam of incident light L1 of a LED
light source 11. The light source 11 can also be a laser diode or
Super Luminescent Diode (SLED) or another light source 11. The
incident light beam L1 is reflected under total internal reflection
conditions at the reference region 120 and, in the absence of an
influencing target substance (2), also at the investigation region
113 to form a light beam L2. The light beam L2 is detected at a
detector 18 to determine the intensity of light reflected at the
reference region 120 and investigation region 113, respectively. A
calibrator 20 compares the outgoing light L2 of both the reference
region 120 and the investigation region 113 as detailed in the
discussion of FIG. 1, and as further detailed in the discussion of
FIG. 12.
[0095] FIGS. 6 and 7 show schematically and exemplarily a top view
and side view, respectively, of an embodiment of a sensing device
100. The sensing device 100 comprises a carrier 110. The carrier
110 has a sensing surface 112. On the sensing surface 112, a fluid
delimiter 101 is positioned such as to allow a preferably liquid
medium 4 to be added to the sensing surface 112. The fluid
delimiter 101 has openings forming a fluid channel system
comprising a fluid reception opening 112, a channel leading to a
measurement chamber, a channel leading away from the reference
chamber, and a vent opening 119. The measurement chamber comprises
an investigation region 113 of the sensing surface 112. Adjacent to
the measurement chamber are two reference regions 120, each
comprising a solid reference element to allow total internal
reflection of incident light in the carrier 110.
[0096] In use, the investigation region 113 and one or both of the
reference regions 120 are irradiated by an incident light beam (not
shown) in the manner depicted in FIG. 1. The light is reflected
under total internal reflection conditions at the reference
region(s) 120 and/or the investigation region 113. A fluid medium
4, preferably a liquid, is added to the sensing device 100 via
fluid reception opening 120. Air contained in the measurement
chamber and channel leading thereto is expelled via vent opening
119. The medium 4 is channeled to the measurement chamber. In the
measurement chamber, a target substance 2 can influence the
intensity of light reflected at the investigation region 113 of the
sensing surface 112 under total internal reflection conditions.
[0097] The intensities of light reflected under total internal
reflection conditions at the investigation region 113 and the
reference region(s) 120 are detected by a detector 18. A calibrator
20 compares the outgoing light of both the reference region(s) 120
and the investigation region 113 in the manner described for FIG.
1.
[0098] FIG. 8 shows schematically and exemplarily a graph of a
detection signal and a reference signal of a device of FIGS. 6 and
7. A normalized signal intensity s.sub.n is graphed. As can be
seen, the characteristic signal 220 obtained from light reflected
at a reference region 120 of the sensing surface 112 shows some
drift in signal intensity. This detected drift in signal intensity
can be used to correct and calibrate the characteristic signal 213
obtained from light reflected at the investigation region 113 of
the sensing surface 112.
[0099] FIGS. 9 and 10 show schematically and exemplarily a side
view and top view, respectively, of a further embodiment of a
sensing device 100. The device 100 comprises a carrier 110 having a
sensing surface 112 thereon. The sensing surface 112 comprises a
series of recesses to form a series of lowered reference regions
120 therein. The recesses may be formed by surface patterning, e.g.
by using focus ion beam milling or pulsed laser ablation. Adjacent
to the reference areas 120 is an investigation region 113 as
described with regards to FIG. 1.
[0100] In use, a liquid medium 4 passes over the sensing surface
112 of the carrier 110. For purposes of illustration, only a
droplet of medium 4 is shown in FIGS. 9 and 10. The medium 4 cannot
enter the recesses at the reference region 120 due to its surface
tension, entrapping air between the medium droplet 4 and the
reference region 120. The air then functions as reference element
121 as described regarding FIG. 1.
[0101] Furthermore, also modifications could be made to a top
fluidic part of a device of the type depicted in FIGS. 6 and 7 such
as to cause air bubbles to be trapped at predefined areas,
preferable inside or close to the fluidic channel. Such trapped air
bubbles would then function as reference element 121 as described
regarding FIG. 1.
[0102] FIG. 11 shows schematically and exemplarily a side view of a
further embodiment of a sensing device 100. The device comprises a
carrier 110 having a sensing surface 112 formed thereon. The
sensing surface 112 comprises an investigation region 113 as
described for FIG. 1, and a reference region 120. The sensing
surface 112 of the reference region 120 is tilted relative to the
sensing surface 112 of the investigation region 113 by an angle of
.beta.. Parallel incident light beams L1 reaching both the
investigation region 113 and the reference region 120 will thus
effectively reach the reference region 120 at an angle of
.theta..sub.crit+.beta.+.delta. instead of .theta..sub.crit+.delta.
as for the angle of incidence at the investigation region 113. The
constraints regarding the refractive index n.sub.TWR of the
reference element 121 is then relaxed to approximately
n TWR < n 2 + ( .beta. + .delta. ) 1 - n 2 2 n 1 2 ( 3 )
##EQU00002##
[0103] where n.sub.2 is the expected refractive index of a medium 4
and n.sub.1 is the refractive index of the carrier 110 material.
Typically, under such relaxed conditions it is easier to find a
suitable material for a reference element 121.
[0104] FIG. 12 shows schematically and exemplarily a side view of
an embodiment of an analyzer device 10. A sensing device 100 has
been inserted in the analyzer device 10. The sensing device 100
comprises, in this embodiment, a carrier 110 having a sensing
surface 112 thereon. Further, a fluid delimiter 101 is positioned
on the carrier 110. A top fluidic part 14 completes a measuring
chamber between the sensing surface 112, the fluid delimiter 101
and the top fluidic part 14.
[0105] The analyzer device 10 further comprises a magnetic element
13 which provides a magnetic field for forcing magnetic particles 2
onto the sensing surface 112 of the sensing device. The magnetic
particles 2 are detected by, in this embodiment, illuminating the
sensing surface 112 with a light beam L1 generated by a light
source 11. The light source 11 is, for example, a laser device,
SLED or a LED. Light L2 reflected from the sensing surface 112 is
detected by a detector 18. The detector 18 is, for example, a
photodetector or a two-dimensional camera. Optical elements can be
arranged in the light beams L1 and L2 for generating parallel light
beams L1 and L2, respectively. Such optical elements are preferably
lenses.
[0106] The carrier 110 is accommodated in an analysis region. In
the analysis region, both incident light L1 can be directed at the
reference region 120 and the investigation region 113 such that
light reflected under total internal reflection conditions can be
detected by the detector 18. Furthermore, at the analysis region
the magnetic field created by magnetic element 13 can force
magnetic particles 2 onto the sensing surface 112.
[0107] The sensing surface 112 further comprises a reference region
120 and an investigation region 113; both are not shown. Incident
light L1 arriving at the reference region 120 and investigation
region 113 of the sensing surface 112 is reflected under total
internal reflection conditions to become a light beam L2. However,
at the investigation region 113 such internal reflection may be
under frustrated total internal reflection conditions. That is to
say that upon movement of magnetic particles 2 onto the
investigation region 113, evanescent light present thereat due to
the incident light beam L1 is scattered and absorbed, resulting in
a change of intensity of light L2 reflected off the investigation
region 113. This change can be detected by a detector 18 as
described above.
[0108] The detector 18 yields two characteristic signals 213, 220.
The intensity of light reflected under total internal reflection
conditions at the investigation region 113 is yielded as first
characteristic signal 213, the intensity of light reflected under
total internal reflection conditions at the reference region 120 is
yielded as second characteristic signal 220. A calibrator 20 then
compares the first characteristic signal 213 and the second
characteristic signal 220 to produce a corrected result. The result
of the comparison is displayed by a display device 21.
[0109] FIG. 13 shows schematically and exemplarily magnetic beads
bound to an investigation region 113 of a sensing device. An
investigation region 113 of a sensing surface 112 is coated with
antibodies 114. The antibodies 114 can bind an analyte 2'. Further,
magnetic beads 2 are coated with antibodies to bind analyte 2'.
Upon addition of analyte 2' in a medium 4, analyte 2' is sandwiched
between antibodies 114 and magnetic beads 2, thus effectively
coupling the magnetic beads 2 to the investigation region 113. The
magnetic beads 2 can then influence the light reflected at the
investigation region 113 as described with respect e.g. to FIG. 12.
The magnetic beads are preferably particles having at least one
dimension ranging between 3 nm and 10000 nm, preferably between 10
nm and 3000 nm, and more preferably between 200 nm and 1000 nm.
[0110] FIG. 14 shows schematically and exemplarily a method of the
present invention. In step 401, a sensing surface 112 with an
investigation region 113 and reference region 120 is provided. A
reference element 121 is located at the reference region 120 to
shield the reference region 120 such that light reflected at the
reference region 120 under total internal reflection conditions
remains unaffected by the presence or absence of a target substance
2 or coloured particulates of a medium 4 comprising the target
substance 2 and, where applicable, an analyte. Providing such
reference element 121 at the reference region 120 may in some
embodiments be performed as an independent method step.
[0111] After step 401, the investigation region 113 and the
reference region 120 are illuminated under total internal
reflection conditions in step 402. A detector 18 detects light
reflected from the investigation region 113 (step 403) and the
reference region 120 (step 404) under total internal reflection
conditions. Steps 403 and 404 can be performed simultaneously, e.g.
using a CCD detector. A first characteristic signal 213 is obtained
from the detector 18 depending on the light reflected from the
investigation region 113. A second characteristic signal 220 is
obtained from the detector 18 depending on the light reflected from
the reference region 120.
[0112] In a further step 410, the first 213 and second 220
characteristic signals are read by a calibrator 20. The calibrator
20 calibrates the first characteristic signal 213 in view of the
second characteristic signal 220. A calibrated first characteristic
signal 213 is output by the calibrator 20.
[0113] In the above described embodiments, the medium 4 was
preferably blood. In other embodiments, the fluid can be any other
fluid, in particular any other body fluid, for example saliva or
urine. The preferred application of the sensing and analyzer device
10 is in the field of point of care diagnostics and detection of
drugs or the detection of the occurrence of a heart attack, and in
particular based on finger prick blood samples and saliva
samples.
[0114] The device, methods and systems of this invention are suited
for sensor multiplexing (i.e. the parallel use of different sensors
and sensor surfaces), label multiplexing (i.e. the parallel use of
different types of labels as target substances 2) and chamber
multiplexing (i.e. the parallel use of different reaction
chambers).
[0115] The devices and methods described in the present invention
can be used as rapid, robust, and easy to use point-of-care
biosensors for small sample volumes. The carrier can be a
disposable item to be used with a compact reader, containing one or
more magnetic field generating means and one or more detection
means. Also, the devices and methods of the present invention can
be used in automated high-throughput testing. In this case, the
carrier is e.g. a well plate or cuvette type carrier, fitting into
an automated instrument.
[0116] In the above embodiments, the device uses evanescent field
techniques for determining the amount of magnetic particles on the
investigation region 113 of the sensing surface 112. In other
embodiments, other substances may be detected. In addition to
molecular assays, also larger moieties can be detected, e.g. cells,
viruses, or fractions of cells or viruses, tissue extracts and so
on.
[0117] In particular, the embodiments have been described with
reference to a sandwich assay as described in FIG. 13. However,
other assay types can be employed, e.g. binding/unbinding assay,
sandwich assay, competition assay, displacement assay, enzymatic
assay, etc.
[0118] The target substance 2 may be directly analyzed by its
presence on the investigation region 113 without use of magnetic
beads. This is particularly preferred for target substances 2 with
a high ability to absorb or scatter light of an evanescent field on
the investigation region 113. In addition or alternatively thereto,
the target substance 2 can be further processed prior to detection.
An example of further processing is that further materials are
added or that the (bio)chemical or physical properties of the
target substance 2 are modified to facilitate detection. The target
substance 2 may for example be reacted with a reactant to alter its
ability to absorb or scatter light of an evanescent field on the
investigation region 113. In particular, the target substance 2 may
be covalently linked to a light absorbing or scattering substance
to influence an evanescent field in the investigation region
113.
[0119] The detection can occur with or without scanning of the
sensor element with respect to the sensing surface 112, in
particular the reference region 120 and/or the investigation region
113.
[0120] In the above embodiments, the reference element 121 has been
described as being an element transparent to incident light but
chosen to effect reflection under total internal reflection
conditions. However, the reference element 121 may also be a true
mirror 121, preferably a metal layer, to reflect incident light.
This abolishes the need to select such materials for the
manufacture of the reference element 121 that have a sufficient
refractive index in view of the refractive index of the sensing
surface material and the incident light.
[0121] Other variations to the disclosed embodiments can be
understood and effected by those skilled in the art in practicing
the claimed invention, from a study of the drawings, the disclosure
and the appended claims.
[0122] In the claims, the word "comprising" does not exclude other
elements or steps, and the indefinite particle "a" and "an" does
not exclude a plurality.
[0123] A single unit or device may fulfill the functions of several
items recited in the claims. The mere fact that certain measures
are recited in mutually different dependent claims does not
indicate that a combination of these measures cannot be used to
advantage.
[0124] Any reference signs in the claims shall not be construed as
limiting the claim's scope. The figures and embodiments shall not
be construed as limiting the claims' scope.
* * * * *